Summary/Abstract Mass spectrometry (MS) has played a leading role in the past three decades in the field of proteomics. A combination of innovative MS-based techniques has provided powerful tools for proteomic discovery including the ability to identify protein biomarkers in a complex sample, quantify changes in protein expression and characterize protein- protein interactions. A second important advance in MS has been the introduction of mass spectrometric imaging (MSI) which extends MS to the spatial dimension, allowing mapping of the distribution of biomolecules including proteins, nucleic acids, metabolites and even small drug compounds in complex tissues. The goal of this Phase II project is to further develop the ability of MSI to perform highly multiplexed imaging, even on the subcellular nanoscale, of targeted biomolecules in biospecimens. Such a capability would provide a major tool for systems biologists and cancer researchers, who require a detailed knowledge of the distribution of key molecules in complex tissues at the cellular, subcellular and molecular levels. It would also provide pathologists with a powerful new tool to analyze tumor tissue specimens in order to ultimately obtain improved therapy and patient outcomes. During Phase I we have successfully demonstrated: i) the ability to simultaneously image by MSI potentially hundreds of targeted biomarkers from formalin fixed paraffin embedded (FFPE) thin sections from mouse brain using proprietary improved photocleavable mass-tags (iPC-MTs) which are incorporated into antibodies or oligonucleotide probes. In contrast, conventional light microscopy-based immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods can image only a few targeted biomolecules; and ii) designed new iPC-MT-probes which are fully compatible with the new method of expansion microscopy (ExM) to obtain nanoscale subcellular MSI resolution. During Phase II, we will build on this progress by applying iPC-MT-probe technology to the analysis of archived breast cancer FFPE specimens in order to ultimately achieve improved cancer histopathology for routine clinical practice. As a model system, 10 iPC-MT-antibody probes and 10 iPC-MT- oligonucleotide probes targeted at specific breast cancer tumor antigens and miRNA biomarkers will be developed and initially tested for potential cross-reactivity using AmberGen's proprietary Bead-Array Mass Spectrometry technology (Bead-AMS?). Breast cancer tissue slices will then be analyzed by MSI using these probes and compared to results obtained from conventional fluorescence based IHC and FISH methods. In order to obtain increased spatial resolution, iPC-MT-probes that cross-link with or are delivered to expanded acrylate gels containing expanded FFPE slices will be used. MSI of the expanded gels will be achieved using specialized low- temperature, infrared laser-based MALDI-MSI instrumentation. This work will be facilitated by our continued collaboration with leading experts in the MS and ExM fields including Drs. Cathy Costello (BU, William Fairfield Warren Distinguished Professor, Director of BU Center for Biomedical Mass Spectrometry), Ed Boyden (MIT, Y. Eva Tan Professor of Neurotechnology) and Jason Amsden (Duke University, Assistant Research Professor).